The present invention relates to a DNA fragment containing a nucleotide sequence encoding esterase which is highly heat stable and asymmetrically hydrolyzes carboxylic acid esters, an esterase encoded by the DNA fragment, a recombinant plasmid containing the DNA fragment, a microorganism transformed with the recombinant plasmid and methods of producing optically active carboxylic acids and their enantiomeric esters.
An optically active carboxylic acid and its enantiomeric ester, which are represented by the general formula (II); ##STR2## (wherein R.sub.1 is alkyl, aralkyl or aryl, R.sub.2 is alkyl, and n is 1 or 2), are useful as a raw material of various physiologically active substances.
The present inventors disclosed methods of asymmetrically hydrolyzing a racemic mixture of carboxylic acids esters to produce an optically active carboxylic acid and its enantiomeric ester using an enzyme or a microorganism, the carboxylic acid esters being represented by the general formula (I); ##STR3## (wherein R.sub.1, R.sub.2 and n are the same meaning as described above and R.sub.3 is alkyl) [see Japanese Patent Application KOKAI No. 12992/1985, No. 12993/1985].
The present inventors isolated Pseudomonas putida (FERM BP-3846) from a soil and found that it produces esterase having a potent, asymmetrical hydrolysis activity for a racemic mixture of carboxylic acid esters, and disclosed the bacterium in Japanese Patent Application KOKAI No. 222798/1989).
To obtain a microorganism having an enhanced enzymatic activity, the recombinant DNA technology is often utilized these days. There has been a method known in the art in which a transformed microorganism is used to increase the production of esterase involved in hydrolysis reaction: a DNA fragment encoding an esterase gene derived from Pseudomonas fluorescens IF03018 is prepared; a host microorganism is transformed with a plasmid having the DNA fragment; esterase is produced from the transformed microorganism (Japanese Patent Application KOKAI No. 67190/1989).
Typically, bacterial cells are directly utilized as "enzyme" rather than utilizing an enzyme purified by a complicated manipulation such as isolation and purification when carboxylic acid esters of the formula (I) are required to be asymmetrically hydrolyzed. In such a method, an efficient reaction relies on several factors such as a potent enzyme activity of bacterial cells, the property of an enzyme that serves to produce carboxylic acids with high optical purity and stability in various conditions such as temperature, pH and the like.
Heat labile enzymes are susceptible to thermal inactivation as reaction time progresses so that it is not easy to increase a reaction rate by raising reaction temperature or to reuse recovered enzyme. Bacterial cells that produce enzyme, which has an excellent heat stability and potent enzymatic activity, are necessary for efficient reaction.
The recombinant DNA technology is an effective method of increasing the enzymatic activity of enzyme produced by bacteria as described in Japanese Patent Application KOKAI No. 67190/1989. The properties of enzyme essentially depends on a DNA sequence that encodes an enzyme. To obtain a highly stable enzyme that satisfies the above requirement, one needs to find a DNA fragment encoding the enzyme.
The Japanese Patent Application KOKAI No. 67190/1989 discloses a microorganism transformed with a recombinant plasmid containing a DNA fragment. The DNA fragment comprises the sequence encoding an esterase gene derived from Pseudomonas fluorescens IF03018. Esterase produced by the microorganism is less heat stable and is inactivated in several hours, for example, at 45.degree. C. or more.
It is a primary object of the present invention to prepare a DNA fragment encoding a highly heat stable esterase for the asymmetrically hydrolysis of carboxylic acid esters of the formula (I) and to efficiently carry out the asymmetrical hydrolysis using a transformant that contains the DNA fragment and efficiently produces the enzyme.
The present inventors have investigated an efficient method of asymmetrically hydrolyzing carboxylic acid esters of the formula (I) using enzyme. We have successfully found that a microorganism transformed with a recombinant plasmid containing an esterase gene derived from Pseudomonas putida (FERM BP-3846), which is isolated from a soil, is useful for obtaining a high yield of reaction products and for carrying out an efficient reaction in a short period of time. The microorganism produces enzyme having potent enzymatic activity and the enzyme is not inactivated with time even at a reaction temperature of 50.degree. C. or more.
In addition, we have isolated a highly heat stable, novel esterase substantially in a pure form from the transformed microorganism and successfully characterized the physical and chemical properties of the enzyme.